High efficacy of Azacitidine plus HAG in acute myeloid leukemia: an open-label, single-arm, multi-center, phase 2 study

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy with high mortality and poor outcome, especially for elderly/un ﬁ t (age ≥ 60 years or un ﬁ t patients who are ineligible to receive intensive chemotherapy) with adverse genetic and molecular abnormalities in the newly diagnosed (ND) and refractory/relapsed (R/R) patients. Azacitidine (Aza), a hypomethylating agent, targets epigenetic gene silencing by inhibiting gene expression against malignant phenotypes. The complete remission (CR) rates of Aza combined regimens varied with different drugs, such as, Aza + Midostaurin (20.8%), Aza + Durvalumab (31.3%), Aza + Pracinostat (46%), Aza + Venetoclax (66.4%) in ND AML and Aza + Midostaurin (21.4%), Aza + Nivolumab (22%), Aza + Venetoclax (37.1%) in R/R AML [1 – 7]. Homoharringtonine (HHT) is a natural plant alkaloid isolated from Cephalotaxus and exhibits an anti-leukemic effect [8, 9]. Several studies revealed that HHT- combined regimens are effective and safe in patients with chronic myeloid leukemia of ND or failed prior therapy (imatinib, IFNa) [10 – 13]. It is reported that HHT in combination with cytarabine, aclarubicin/daunorubi- cin (HAA/HDA) achieved a higher CR than the standard “ 3 + 7 ” regimen (DA) in treating de novo AML [8]. HAG (HHT, low-dose cytarabine,


Clinical endpoint and assessments
The primary endpoint was the composite complete remission (complete remission [CR] or complete remission with incomplete hematologic recovery [CRi] ). Secondary endpoints were overall survival (OS) defined as the time from study entry to death from any cause), relapsefree survival (RFS) is defined as the time from achieving a CR until disease recurrence or death), adverse events (AE) (including hematological and non-hematological AE defined as any unfavorable and unintended signs including an abnormal laboratory finding, symptom, or disease). Treatment failure was defined as not achieving CR or CRi after two cycles of induction therapy.
Prespecified correlative assessments included targeted gene panel sequencing to assess associations between somatic mutation patterns and therapeutic responses as well as disease progression.
Response to therapy was monitored by analysis of blood and bone marrow aspirates.
Response assessment was done at the end of cycles 1 and 2 (no CR/CRi after cycle 1), and then after every 2 cycles of consolidation and every 3 months during maintenance to confirm ongoing response. Responses were categorized based on the revised International Working Group criteria for AML[1, 2].
Minimal residual disease (MRD) assessment by multicolor flow cytometry (MFC) was done on pretreatment bone marrow and all subsequent bone marrow examinations with the assay sensitivity of 0.01% as previously described [3]. Bone marrow samples were obtained at diagnosis and evaluated for the presence of cytogenetic and molecular aberrations. For documentation of mutations, the entire coding sequences of 58 genes known to be frequently mutated in myeloid malignancies were sequenced with a targeted leukemia exome-seq panel (

Sample size estimation
In this trial, the optimal Simon's two-stage design method was used to determine the sample size. A composite complete remission rate ≤ 40% (p0) would be considered a null hypothesis (the outcome was unacceptable). While a composite complete remission rate of higher than 60% (p1) would grant the regimen for further exploration.
Baseline demographic and disease characteristics were generally balanced between 40 ND AML patients with Aza+ HAG regimen versus 14 ND AML patients with HAG regimen in the same center (Zhongda Hospital) ( Table S2).
Comparison of CR/CRi rate between Aza+HAG and HAG group was performed using the Chi-square test or Fisher exact test (when the sample size is small and with less degree of freedom). The comparison of OS and RFS between the two groups was performed using Kaplan-Meier estimates with the log-rank test. A threshold P-value < 0.05 was considered a statistically significant difference.

Targeted exome-seq panel for gene mutation screening in AML patients
A leukemia targeted-exome-seq panel including 58 genes was used for screening the gene mutations in 103 enrolled patients by next-generation sequencing (NGS) before and after the induction therapy [4,5]. The target genes in the panel are listed in Table S5.

Agilent SureSelect Human All Exon V4+UTRs (Agilent) was used for the coding exons plus
UTRs of target genes. Probes for each exon of each target gene are designed on NCBI (https://www.ncbi.nlm.nih.gov/). The targeted exome-seq method is performed as reported [6].
Briefly, the genomic DNA was isolated from bone marrow samples with the genomic DNA isolation kit (Qiagen, Hilden, Germany). All DNA samples were sheared with a Covaris E220 instrument generating approximately 260 bp DNA fragments. The fragmented DNA was processed into Illumina-compatible sequencing libraries using Kapa Hyper Prep Kit (Illumina, San Diego, CA, USA). Each library was uniquely barcoded and captured by the leukemia panel probes, followed by PCR amplification and sequencing on a HiSeq 2500 (Illumina) with 2x100 bp reads. The sequencing reads were aligned to the human genome by following Broad Institute's GATK best-practice pipeline to call germline short variants (SNPs and Indels). Called variants were annotated using ANNOVAR (version 2.3). Exonic variants with exonic, nonsynonymous, stop-gain, or stop-loss, novel SNPs, and with predicted deleterious/damaging functions were manually surveyed by IGV to confirm.
The association of gene mutations with clinical response, relapse, and risk status was analyzed with R 4.0.1 software and depicted as a waterfall figure. The association of the gene mutations with OS and RFS was also evaluated by the Kaplan-Meier method [7][8][9].

Meta-analysis of HAG regimen in treating elderly AML patients
To evaluate the clinical response of the HAG regimen in treating an elderly patient with AML, we conducted a meta-analysis of the HAG regimen by carefully screening MEDLINE, PubMed, EMBASE, and CNKI (Chinese) databases. Inclusion criteria were: 1) included unfit AML (previously untreated) patients (age over 60 years or ineligible for receiving standard chemotherapy) who received HAG regimen; 2) reported the clinical responses (CR or CRi).

Statistical analysis
The distribution of survival was estimated with the use of the Kaplan-Meier method. The lower limit and upper limit of 95% confidence interval were calculated by the Wilson method.
The student t-test was used to identify differences between groups. Categorical parameters were compared with the chi-squared test or Fisher's exact test. Statistical analysis was performed on STATA 16.0 software.

Data sharing statement
The patient datasets for the current study are not publicly accessible following local health research ethics protocols; however, they may be available from the corresponding author. De-identified individual-level data and the data dictionary will be made available to qualified researchers who present study protocols, which will require approval by the institute health research ethics committee and principal investigator. These data will only be made available from study sites at which the institution and ethics review board allow such release.  Tables   Table S1 Baseline Table S5 The Leukemia Panel for next generation sequencing   ABL1  BRAF  CEBPA  ETV6  HRAS  ANKRD26  CALR  CSF3R  EZH2  IDH1  ASXL1  CBL  CUX1  FLT3  IDH2  ATRX  CBLB  DDX41  GATA1  IKZF1  BCOR  CBLC  DNMT3A  GATA2  JAK2  BCORL1  CDKN2A  ETNK1  GNAS  JAK3  KDM6A  NPM1  PTEN  SMC1A  TP53  KIT  NRAS  PTPN11  SMC3  U2AF1  KMT2A  PDGFRA  RAD21  SRSF2  WT1  KRAS  PHF6  RUNX1  STAG1  ZRSR2  MPL  PIGA  SETBP1  STAG2  NF1 PPM1D SF3B1 TET2

Fig S1
Clinical procedure of Aza +HAG regimen in this trial (A), a total of 115 AML patients were screened, 3 of them were not stratified according to the inclusion criteria. Finally, 112 patients were enrolled in this trial. Patients withdrawn from the cohort were followed for survival; OS (B) and RFS (C) curve of newly diagnosed AML patients versus secondary AML patients (arising from preexisting myeloid neoplasms).